Oxidative cell wall damage mediated by bleomycin-Fe(II) in Saccharomyces cerevisiae

Lim, Seung Taik; Jue, Chong K.; Moore, Carol W.; Lipke, Peter N.

doi:10.1128/jb.177.12.3534-3539.1995

Cited by 26 publications

(27 citation statements)

References 42 publications

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“…This is the first report of a chemical or drug that blocks fungal septum formation and cytokinesis and that also preferentially localizes to cell walls (35) and alters intact cell walls on nongrowing cells (2,23,32,35,40) and isolated cell walls (2). In this regard, the bleomycin drug family represents a previously unknown class of cidal antifungal agents.…”

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

“…The discovery that the fungal cell wall is a target of a family of oxidative agents widely used for anticancer chemotherapy was made in S. cerevisiae (35). The mechanism of action of the low-molecular-weight bleomycins (M r approximately 1,500 to 1,600 [57,58]), structurally related metalloglycopeptidic antibiotics (5), involves an initial localization (35) and subsequent injury to cell walls (23,32,35), altered anchorage of mannoproteins to the cell wall matrix (2), and the release of specific species of surface mannoproteins from intact as well as isolated cell walls (2).For the present studies, we investigated the effects of the metalloglycopeptides on fungal cell walls, cell growth, cell wall septation, and cell division. We reasoned that these basic cellular processes could be at risk, since a specific target of the bleomycin drug family is the fungal cell wall (35).…”

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Fungal Cell Wall Septation and Cytokinesis Are Inhibited by Bleomycins

Moore

McKoy

Valle

et al. 2003

Antimicrob Agents Chemother

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When the essential and distinctive cell walls of either pathogenic or nonpathogenic fungi break, cytoplasmic membranes rupture and fungi die. This fungicidal activity was discovered previously on nonproliferating Saccharomyces cerevisiae cells treated briefly with the oxidative tool and anticancer drug family of bleomycins. The present studies investigated effects of bleomycin on growing fungal organisms. These included the medically important Aspergillus fumigatus and Cryptococcus neoformans, as well as the emerging human pathogen and fungal model, S. cerevisiae. Bleomycin had its highest potency against A. fumigatus. Scanning electron microscopy and thin-section transmission electron microscopy were used to study morphological growth characteristics. Killing and growth inhibition were also measured. Long, thin, and segmented hyphae were observed when A. fumigatus was grown without bleomycin but were never observed when the mold was grown with the drug. Bleomycin arrested conidial germination, hyphal development, and the progression and completion of cell wall septation. Similarly, the drug inhibited the construction of yeast cell wall septa, preventing cytokinesis and progression in the cell division cycle of S. cerevisiae. Even when cytoplasms of mother and daughter cells separated, septation and cell division did not necessarily occur. Bizarre cell configurations, abnormally thickened cell walls at mother-daughter necks, abnormal polarized growth, large undivided cells, fragmented cells, and empty cell ghosts were also produced. This is the first report of a fungicidal agent that arrests fungal growth and development, septum formation, and cytokinesis and that also preferentially localizes to cell walls and alters isolated cell walls as well as intact cell walls on nongrowing cells.The unique cell wall that surrounds fungal cells is absolutely essential for their survival. The cell wall of pathogenic fungi has evolved for survival from environmental factors and host defenses (7,16,18,21,60). The ability of cell walls to evade such factors contributes to the power of pathogenic fungi to cause a wide spectrum of clinical disease (45). Moreover, the unique arrangement of the molecular species forming the structure of the fungal cell wall makes it an excellent target for drug intervention (3,6,24,44,46,54). Thus, the elucidation of the mechanisms of action of agents that interact with the molecules forming the cell walls will yield new insights into fungal cell wall structure, formation of cell walls between dividing cells, and cell division. For fungal pathogens, an understanding of the mechanisms of action of cell wall-specific agents should assist in the design of wall-specific antifungal drugs.Saccharomyces cerevisiae has long been a unicellular fungal model for studies of basic mechanisms. Since this budding yeast is also an emerging opportunistic human pathogen (15,38,42) with properties frequently observed in pathogenic fungi (4,29,30), the yeast is also developing as a model fungal pathogen (12). The dis...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Fungal Cell Wall Septation and Cytokinesis Are Inhibited by Bleomycins

Moore

McKoy

Valle

et al. 2003

Antimicrob Agents Chemother

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“…S. cerevisiae Sc377 was transformed with pWZ1-6, pWZ1-6a, or pVTU102. Bleomycin sensitivity was determined essentially by the method of Lim et al (19); cisplatin sensitivity was determined by the method of McA'Nulty and Lippard (22).…”

Section: Methodsmentioning

confidence: 99%

The Nucleic Acid Binding Activity of Bleomycin Hydrolase Is Involved in Bleomycin Detoxification

Zheng

Johnston

1998

Molecular and Cellular Biology

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Yeast bleomycin hydrolase, Gal6p, is a cysteine peptidase that detoxifies the anticancer drug bleomycin. Gal6p is a dual-function protein capable of both nucleic acid binding and peptide cleavage. We now demonstrate that Gal6p exhibits sequence-independent, high-affinity binding to single-stranded DNA, nicked doublestranded DNA, and RNA. A region of the protein that is involved in binding both RNA and DNA substrates is delineated. Immunolocalization reveals that the Gal6 protein is chiefly cytoplasmic and thus may be involved in binding cellular RNAs. Variant Gal6 proteins that fail to bind nucleic acid also exhibit reduced ability to protect cells from bleomycin toxicity, suggesting that the nucleic acid binding activity of Gal6p is important in bleomycin detoxification and may be involved in its normal biological functions.Bleomycin hydrolase is a cysteine peptidase that can cleave the glycopeptide bleomycin, which has been used widely as an anticancer drug in chemotherapy. Bleomycin hydrolase is highly conserved in organisms ranging from bacteria to humans (3, 12). The yeast form of this enzyme has been purified, and its crystal structure has been solved (12). We designated the yeast form of bleomycin hydrolase Gal6, as its expression is regulated by galactose (12, 35). We first identified Gal6p by virtue of its DNA binding property. It binds single-stranded DNA with high affinity (10 nM) and double-stranded DNA with much lower affinity (1 M) (33).The crystal structure of Gal6p revealed that it is a hexamers, its six subunits forming a ring with a central channel of about 20-Å diameter at the entrance. The peptidase active sites are located deep inside this tunnel, sequestered from the exterior. Other nucleic acid-binding proteins with a ring structure, e.g., Escherichia coli DNA polymerase ␤ subunit (16), eukaryotic proliferating cell nuclear antigen (17), and Bacillus subtilis Trp RNA-binding attenuation protein (2), have been identified, as have other proteases with ring structures, e.g., the proteasome and tricorn, which form a unique self-compartmentalizing protease family (18,21). However, Gal6 is the only ring protein with both of these activities.Although a role for Gal6p in bleomycin detoxification has been well documented (7,13,25,33), there is no evidence to date that its nucleic acid binding activity plays a role in this detoxification. The fact that bleomycin itself cleaves nucleic acids (14,28) suggests that the peptidase and nucleic acid binding activities of Gal6p may be functionally linked. On the other hand, the DNA binding activity of Gal6p could be an in vitro artifact and may not reflect a biologically relevant function.We report here the characterization of the nucleic acid binding activity of Gal6p. It binds to single-stranded DNA as well as single-stranded RNA. Immunolocalization demonstrates that most of the Gal6 protein is located in the cytoplasm, indicating that it may function mainly as an RNA-binding protein. Mutation studies show that the ␤-hairpin structure at the opening of th...

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“…• C. Bleomycin sensitivity was determined by adding bleomycin to YPD plates to different final concentrations, as indicated in the text, and by the method of Lim et al (1995). For plate assay, equal amounts of serial dilutions of cells from exponentially cultures growing in YPD medium were spotted onto plates of the same medium containing either no drug, 2 µg or 5 µg bleomycin/ml.…”

Section: Methodsmentioning

confidence: 99%

MIG1‐dependent and MIG1‐independent regulation of GAL gene expression in Saccharomyces cerevisiae: role of Imp2p

Alberti

Lodi

Ferrero

et al. 2003

Yeast

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Imp2p (Yil154c) is a transcriptional activator involved in glucose derepression of the maltose, galactose and raffinose utilization pathways and in resistance to thermal, oxidative or osmotic stress. We analysed the role of Imp2 in the regulation of GAL genes. Imp2 was shown to have a positive effect on glucose derepression of Leloir pathway genes and their activator gene GAL4. The effect of Imp2 on galactose metabolism was shown to be partially dependent on Mig1p. The Mig1-independent role depends on Nrg1p. However, disruption of both MIG1 and NRG1 only partially relieves the glucose repression of GAL genes in the imp2 mutant, indicating that Imp2 must also have other function(s). Moreover, the interaction between IMP2 and GAL6/BLH1, a recently isolated gene involved in the regulation of GAL genes that shares with Imp2 the ability to protect cells from the glycopeptide bleomycin, was also analysed. The results suggest a major role of Imp2 in a GAL6-independent pathway.

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Oxidative cell wall damage mediated by bleomycin-Fe(II) in Saccharomyces cerevisiae

Abstract: Bleomycin mediates cell wall damage in the yeast

Cited by 26 publications

References 42 publications

Fungal Cell Wall Septation and Cytokinesis Are Inhibited by Bleomycins

Fungal Cell Wall Septation and Cytokinesis Are Inhibited by Bleomycins

The Nucleic Acid Binding Activity of Bleomycin Hydrolase Is Involved in Bleomycin Detoxification

MIG1‐dependent and MIG1‐independent regulation of GAL gene expression in Saccharomyces cerevisiae: role of Imp2p

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